An acid-sensitive polymer containing a beta-carboxyl amide bond, and a method of preparation and use thereof

Abstract

The application belongs to the technical field of medical materials and relates to an acid-sensitive responsive amphiphilic block polymer, wherein a hydrophilic segment is polyethylene glycol, a hydrophobic segment is a homopolymer or copolymer based on a triethylene carbonate derivative, and a side chain carboxyl of the hydrophobic segment is connected to a main chain through a beta-carboxyl amide bond, the beta-carboxyl amide bond has the characteristics of being stable under neutral and alkaline conditions and being broken under acidic conditions. A drug carrier prepared from the acid-sensitive responsive amphiphilic block polymer has excellent loading performance on alkaline polypeptide / protein drugs through electrostatic attraction between the side chain carboxyl of the polymer and the alkaline polypeptide / protein drugs, and the drug loading capacity can reach 1% to 17%. Meanwhile, after the drug carrier is taken into lysosomes by cells, the drug carrier effectively responds to the acidic microenvironment in the lysosomes, undergoes charge reversal, accelerates lysosome escape and intracellular drug release, and enhances the curative effect of the polypeptide / protein drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials. It relates to an acid-sensitive polymer containing a β-carboxyl amide bond and a preparation method and use thereof. Specifically, it relates to the synthesis of an acid-sensitive responsive amphiphilic block polymer based on a trimethylene carbonate derivative, and the application of the polymer as a basic polypeptide / protein drug carrier material, BACKGROUND

[0002] The prior art discloses polypeptide and protein drugs such as antibodies, antigens, growth factors, bioactive peptides, etc., which show great potential in the treatment of various diseases due to their high specificity and activity. However, studies have shown that polypeptide / protein drugs have poorer stability compared to traditional small molecule drugs, and their therapeutic effect may be lost due to aggregation, degradation or structural unfolding. Encapsulation or conjugation of polypeptide / protein drugs inside or on the surface of carriers prepared from specific polymer materials helps to improve the stability of the drugs and enhance the therapeutic effect. Currently commonly used biodegradable polymer carrier materials include polylactic acid (PLA), polyglycolic acid (PGA), lactic acid and glycolic acid copolymer (PLGA), polycaprolactone (PCL), polytrimethylene carbonate (PTMC), polyhydroxybutyrate (PHB), polyglycerol sebacate (PGS) and polylactic acid (PCA), etc. These materials are all lipophilic materials, which are generally connected with hydrophilic segments (such as polyethylene glycol (PEG)) to form amphiphilic block copolymers, and then micelles, nanoparticles, vesicles, etc. are prepared to encapsulate drugs. However, there are great difficulties in using these materials to encapsulate polypeptide / protein drugs, because most polypeptide / protein drugs are highly water-soluble and have poor affinity with the lipophilic core of the nanocarrier, resulting in low polypeptide / protein drug encapsulation efficiency and easy leakage in the body. Even for vesicles with an internal aqueous phase space structure, the effect of loading polypeptide / protein drugs is still not good, and the drug loading capacity is usually less than 1%. Therefore, the development of suitable polymer materials to improve the encapsulation of polypeptide / protein drugs is crucial for their application.

[0003] Poly trimethylene carbonate (PTMC) is a kind of polycarbonate biodegradable polymer, which has good biological safety and is approved by FDA to be used in human body implantation. The amphiphilic block copolymer PEG-PTMC formed by introducing PEG block into PTMC has a wide application in drug delivery. Meanwhile, the monomer TMC of PTMC is easy to be chemically modified to introduce branched structure. A branched PTMC derivative material with acrylic acid structure has been synthesized in the prior art, which reacts with small molecule carboxylic acid containing thiol group to obtain carboxyl-modified PEG-PTMC block copolymer. The polymeric vesicles made of the block copolymer have an encapsulation efficiency of 51% and a drug loading of 10.2% for model protein cytochrome C through electrostatic attraction between carboxyl and amino group of the protein (Li Shaoke, et al. European Journal of Pharmaceutics and Biopharmaceutics, 2012, 82(1), 103-111.). The above results show that enhancing the electrostatic attraction between the polymeric material and the polypeptide / protein drug helps to improve the encapsulation of the nanocarrier for the polypeptide / protein drug. However, the carboxyl group in the material is connected to the polymer backbone through a thioether bond, which is stable under acidic conditions and has no acid-sensitive cleavage, so it is unknown whether the carrier can effectively escape from the lysosome and release the drug in the cell.

[0004] After being taken up by cells, the nanocarrier is transported to the lysosome. The interior of the lysosome is an acidic microenvironment with a pH of about 4.5, and there are various lipases, protein hydrolytic enzymes, etc. If the carrier is degraded and destroyed by the lysosome, the polypeptide / protein drug is easy to be deprotected and hydrolyzed to be ineffective. Therefore, if the nanocarrier can successfully escape from the lysosome and release the drug in the cytoplasm, it is helpful for the polypeptide / protein drug to better exert its efficacy. Studies have shown that when the nanocarrier is positively charged, it can interact with the negatively charged lysosome membrane, causing the lysosome membrane to rupture, thereby causing lysosomal escape. The β-carboxy amide bond is formed by the reaction of a special anhydride with a primary amine group, which has an acid-sensitive response characteristic. It can undergo autocatalytic hydrolysis cleavage under weak acidic conditions (pH 4.5-6.8) to remove the carboxyl group and expose the amino group. Therefore, if the β-carboxy amide bond can be introduced into the side chain of the PTMC derivative material, the carrier formed by the material can remain stable under neutral pH conditions, and through the electrostatic attraction between the multiple carboxyl groups on the polymer branched chain and the amino groups of the polypeptide / protein drug rich in basic amino acids, the encapsulation efficiency and drug loading of the polypeptide / protein drug can be improved. After being taken up by cells, the carrier can undergo β-carboxy amide bond cleavage under the slightly acidic conditions in the lysosome to expose the amino group and destroy the lysosome membrane, which is conducive to the accelerated escape of the carrier from the lysosome and the release of the polypeptide / protein drug encapsulated therein.

[0005] Based on the status quo of the prior art, the inventors of the present application propose an acid-sensitive polymer containing a beta-carboxyl amide bond and a preparation method and use thereof. SUMMARY

[0006] The present application aims to provide an acid-sensitive polymer containing a beta-carboxyl amide bond and a preparation method and use thereof based on the status quo of the prior art

[0007] The present application provides an acid-sensitive responsive amphiphilic block polymer based on a trimethylene carbonate derivative, wherein different degrees of substitution of carboxyl groups are introduced through beta-carboxyl amide bonds in the side chains of the lipophilic segments, so as to improve the encapsulation efficiency of the nano-carrier for basic polypeptide / protein drugs through electrostatic attraction. At the same time, the beta-carboxyl amide bond is hydrolyzed and broken to remove the carboxyl group under acidic pH conditions, exposing the amino group, so that the carrier undergoes charge reversal, not only facilitating the escape of the nano-carrier from the lysosome, but also enhancing the electrostatic repulsion between the carrier and the polypeptide / protein drug, promoting the rapid and complete release of the drug in the cytoplasm, and significantly improving the drug efficacy.

[0008] The chemical structural formula of the acid-sensitive responsive amphiphilic block polymer according to the present application is as follows:

[0009]

[0010] wherein R1 is selected from one of the following groups:

[0011]

[0012] R2 is selected from one of the following groups:

[0013]

[0014] The acid-sensitive responsive amphiphilic block polymer according to the present application is characterized in that the hydrophobic segment is obtained by self-polymerization of a special trimethylene carbonate derivative monomer or ring-opening copolymerization with other carbonate and cyclic ester monomers, and then chemical modification. The chemical structure of the trimethylene carbonate derivative monomer is as follows:

[0015]

[0016] The other carbonate monomers are selected from trimethylene carbonate and 2,2-dimethyltrimethylene carbonate; and the cyclic ester monomers are selected from caprolactone, lactide and glycolide.

[0017] The preparation method of the acid-sensitive responsive amphiphilic block polymer according to the present application comprises the following steps:

[0018] (1) using polyethylene glycol as an initiator, performing ring-opening copolymerization on the above trimethylene carbonate derivative monomer and other carbonate or cyclic ester monomers to prepare a random copolymer intermediate a, the chemical structural formula of the copolymer intermediate a is as follows:

[0019]

[0020] wherein R1 is selected from one of the following groups:

[0021]

[0022] (2) the copolymer intermediate a is deprotected under the condition of Pd / C / H2 to obtain a copolymer intermediate b, the side chain of the copolymer intermediate b has a primary amine group, the chemical structural formula of the copolymer intermediate b is as follows:

[0023]

[0024] (3) the primary amine group in the side chain of the copolymer intermediate b reacts with an anhydride to form a β-carboxyl amide bond, to obtain an acid-sensitive responsive amphiphilic block polymer, the chemical structural formula of the acid-sensitive responsive amphiphilic block polymer is as follows:

[0025]

[0026] wherein R2 is selected from one of the following groups:

[0027]

[0028] the anhydride is selected from citraconic anhydride, 2,3-dimethyl maleic anhydride, cis-aconitic anhydride, and 1-cyclohexene-1,2-dicarboxylic anhydride.

[0029] In the above technical solution of the present application, by controlling the number of trimethylene carbonate derivative monomers and other carbonate or cyclic ester monomers, acid-sensitive responsive amphiphilic block polymers with different molecular weights can be prepared, wherein in structural formula 1, n is 20-120, x is 50-500, and y is 25-250. In order to regulate the charge density and acid-sensitive responsiveness of the polymer, the ratio of x to y in structural formula 1 is 20:1-1:5.

[0030] The present application also provides a synthesis method of the trimethylene carbonate derivative monomer used in the preparation process of the acid-sensitive responsive block polymer, the synthesis of the monomer includes the following steps:

[0031] (1) 2,2-bis(hydroxymethyl)propionic acid and bis(pentafluorophenyl) carbonate are reacted to prepare intermediate 1, and the reaction process is as follows:

[0032]

[0033] (2) The intermediate 1 is reacted with N-benzyloxy carbonyl ethylenediamine to obtain a trimethylene carbonate derivative monomer, and the reaction process is as follows:

[0034]

[0035] In the preparation process, the technical scheme comprises the following steps:

[0036] (1) 2,2-bis(hydroxymethyl)propionic acid and bis(pentafluorophenyl) carbonate are used as reactants (the molar ratio of the two is 1:2-1:5), anhydrous tetrahydrofuran is used as a solvent, cesium fluoride is used as a catalyst, and the reaction is stirred at room temperature for 12-24 hours to obtain intermediate 1;

[0037] (2) The intermediate 1 is dissolved in anhydrous tetrahydrofuran, and N-benzyloxy carbonyl ethylenediamine (the molar ratio of the two is 3:1-1:3) is used as a catalyst, and the reaction is stirred at room temperature for 12-24 hours to obtain a trimethylene carbonate derivative monomer.

[0038] The method for synthesizing the trimethylene carbonate derivative monomer in the technical scheme has great difference from the method for synthesizing the trimethylene carbonate derivative monomer disclosed in Chinese invention patent CN106543136A. The process for synthesizing the trimethylene carbonate derivative monomer in the CN106543136A patent is that the carboxyl group of 2,2-bis(hydroxymethyl)propionic acid is activated by a carbodiimide and a hydroxybenzotriazole, and then connected with N-benzyloxy carbonyl ethylenediamine to form an intermediate. Then the intermediate is reacted with ethyl chloroformate to form a carbonate ring structure. However, in the present application, bis(pentafluorophenyl) carbonate is used instead of ethyl chloroformate to achieve the effect of carbonate ring formation, and it also has the effect of activating the carboxyl group, so the use of other carboxyl activating agents is avoided, and the reaction components are reduced. More importantly, compared with the highly toxic ethyl chloroformate, bis(pentafluorophenyl) carbonate is a commonly used pharmaceutical intermediate, which does not belong to the control range of the highly toxic chemical catalog, has no reported contact toxicity, has no risk of volatilization and inhalation, and is conducive to the industrialized mass production of the product trimethylene carbonate derivative monomer.

[0039] The acid-sensitive responsive amphiphilic block polymer can be used as a drug carrier for polypeptide / protein drugs. The drug carrier prepared from the acid-sensitive responsive amphiphilic block polymer can form electrostatic attraction with the amino group of the basic polypeptide / protein drug through the multiple carboxyl groups on the side chain, thereby greatly improving the encapsulation efficiency and drug loading of the carrier for the basic polypeptide / protein drug. Specifically, as shown in Examples 7 to 12, the drug loading of the nanoparticles prepared from the PEG-PTMC with no carboxyl group modification in the hydrophobic segment is less than 0.5%, and the encapsulation efficiency is less than 10%. However, the encapsulation efficiency of the nanoparticles prepared from the acid-sensitive responsive amphiphilic block polymer for the basic polypeptide / protein drug is increased to 35% to 90%, and the drug loading is increased to 1% to 17%, which shows excellent drug loading performance.

[0040] The drug carrier prepared from the acid-sensitive responsive amphiphilic block polymer according to the present application can undergo hydrolysis of the beta-carboxyl amide bond in the endosome / lysosome in the cell (pH 4.5) to expose the amino group, reverse the Zeta potential from negative to positive, disturb the lysosome membrane, make the carrier easy to escape from the lysosome, and cause electrostatic repulsion between the carrier and the basic polypeptide / protein drug, thereby accelerating the release of the drug in the cytoplasm to fully exert the drug efficacy. The chemical structure change in the hydrolysis process of the beta-carboxyl amide bond is as follows:

[0041] The acid-sensitive responsive amphiphilic block polymer according to the present application can be used in combination with other synthetic amphiphilic polymers to prepare a drug carrier, and the proportion (mass ratio) of the acid-sensitive responsive amphiphilic block polymer is 50% to 100%. The other synthetic amphiphilic polymers include, but are not limited to, polyethylene glycol-polytrimethylene carbonate (PEG-PTMC), polyethylene glycol-poly(lactic acid) (PEG-PLA), polyethylene glycol-poly(lactic acid-glycolic acid) copolymer (PEG-PLGA), polyethylene glycol-poly(caprolactone) (PEG-PCL), polyethylene glycol-poly(hydroxybutyric acid) (PEG-PHB), polyethylene glycol-poly(glyceryl sebacate) (PEG-PGS), and polyethylene glycol-poly(citric acid) (PEG-PCA).

[0042] The method for loading polypeptide / protein drugs in the drug carrier formed by the acid-sensitive responsive amphiphilic block polymer according to the present application is emulsion-solvent evaporation method, and the specific steps are as follows: first, the acid-sensitive responsive amphiphilic block polymer and other synthetic amphiphilic polymers are dissolved in an organic solvent (the organic solvent is preferably chloroform, dichloromethane or ethyl acetate) as an organic phase. The polypeptide / protein drug is dissolved in water / buffer as an aqueous phase. The aqueous phase is mixed with the organic phase, and a W / O type primary emulsion is formed by ultrasonic treatment with a probe-type ultrasonic cell pulverizer. Then, the W / O type primary emulsion is mixed with a surfactant aqueous solution (the surfactant is preferably sodium cholate or polyvinyl alcohol), and a W / O / W type multiple emulsion is formed by ultrasonic treatment. The surfactant aqueous solution is added for dilution, and the organic solvent is removed by rotary evaporation. The unencapsulated drugs are removed by centrifugation or ultrafiltration, and the drug-loaded nanometer preparation is obtained.

[0043] The polypeptide / protein drug can be loaded by the solvent diffusion method according to the present application, and the specific steps are as follows: the acid-sensitive responsive amphiphilic block polymer, other synthetic amphiphilic polymers and the polypeptide / protein drug are dissolved in an organic solvent (the organic solvent is preferably acetone, methanol, acetonitrile, tetrahydrofuran or dimethyl sulfoxide). The mixture is slowly injected into water / buffer under stirring, and the organic solvent and the unencapsulated drugs are removed by dialysis, and the drug-loaded nanometer preparation is obtained.

[0044] The drug carrier formed by the acid-sensitive responsive amphiphilic block polymer according to the present application has various types, including but not limited to polymer nanoparticles, polymer vesicles, polymer micelles and nanocomposites.

[0045] The beneficial effects of the present application mainly include:

[0046] 1. The acid-sensitive responsive amphiphilic block polymer according to the present application has multiple carboxyl groups in the side chain, which can efficiently encapsulate basic polypeptide / protein drugs by electrostatic attraction. The degree of substitution of the carboxyl groups in the side chain is controllable, and the strength of the carrier-drug interaction can be adjusted as needed to optimize the encapsulation effect.

[0047] 2. The acid-sensitive responsive amphiphilic block polymer according to the present application has a β-carboxyl amide bond between the carboxyl group in the side chain and the polymer main chain, which has acid-sensitive cleavage properties. The drug carrier constructed by the polymer remains stable in the neutral pH of the body circulation, slows down the in vivo clearance of the polypeptide / protein drug, and after being taken up by cells into the weakly acidic environment of lysosomes, the charge is reversed, effectively escaping from the lysosomes, and the drug is released rapidly in the cytoplasm, thereby enhancing the pharmacological effect of the drug.

[0048] 3. The acid-sensitive responsive amphiphilic block polymer according to the present application is composed of polyethylene glycol and trimethylene carbonate derivatives, and the drug carrier formed by the polymer has excellent biocompatibility and biodegradability. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1, PEG-PTMC(DMA) monomer 1 H NMR spectra.

[0050] Figure 2 , PEG-PTMC(Cit) monomer 1 H NMR spectra.

[0051] Figure 3 , PEG-PTMC(SA) monomer 1 H NMR spectra.

[0052] Figure 4 Zeta potential change of PEG-P(CL-co-TMC(Aco)) and PEG-P(CL-co-TMC(GA)) nanoparticles in pH 7.4 and 5.0 buffer.

[0053] Figure 5 Cumulative release profile of NC-1900 loaded PEG-P(CL-co-TMC(Aco)) and PEG-P(CL-co-TMC(GA)) nanoparticles in pH 7.4 and 5.0 buffer.

[0054] Figure 6 Lysosomal escape effect of PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-P(LG-co-GA-co-TMC(SA)) nanoparticles.

[0055] Figure 7 Intracellular drug release of PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-P(LG-co-GA-co-TMC(SA)) nanoparticles.

[0056] Figure 8 Effect of AGA peptide loaded NP(Cit) and NP(SA) nanoparticles on Aβ 25-35 cytotoxicity.

[0057] Figure 9 Stability of SAL loaded nanoparticles (NP(DMA)) prepared from polymer PEG-P(LA-co-TMC(DMA)) and free SAL in plasma and brain homogenate.

[0058] Figure 10 Cytotoxicity of PEG-P(LA-co-TMC(DMA)), PEG-P(CL-co-TMC(Aco)), PEG-P(LG-co-GA-co-TMC(DCA)) blank nanoparticles. DETAILED DESCRIPTION

[0059] The present invention is illustrated by the following description and embodiments. The following description is non-limiting and does not limit the scope of the claims of the present invention.

[0060] Example 1: Synthesis of trimethylene carbonate derivative monomers

[0061] 2,2-bis(hydroxymethyl)propionic acid and bis(pentafluorophenyl) carbonate (3Eq.) were placed in a round-bottom flask and dissolved in anhydrous tetrahydrofuran. Cesium fluoride (3Eq.) was added under stirring, and the reaction was carried out at room temperature for 12–24 h under nitrogen protection. Tetrahydrofuran was removed by rotary evaporation, and dichloromethane was added to dissolve it completely. After standing, the mixture was filtered to remove insoluble byproducts, retaining the organic phase. An equal volume of saturated Na₂CO₃ solution was added to the organic phase, and the mixture was shaken thoroughly and allowed to stand. The aqueous phase was discarded, and the mixture was washed three times. An appropriate amount of anhydrous MgSO₄ powder was added to adsorb moisture, and the mixture was filtered. The resulting organic phase was evaporated to dryness by rotary evaporation, precipitating a white solid. This solid was completely dissolved in sufficient ethyl acetate. Hexane was slowly added dropwise until a large amount of white flaky crystals precipitated. After standing, the mixture was filtered to obtain intermediate 1, with a yield of 65%.

[0062] At room temperature, N-benzyloxycarbonyl ethylenediamine hydrochloride and triethylamine (0.1 Eq.) were dissolved in tetrahydrofuran. Intermediate 1 (1.5 Eq.) was added directly to the reaction solution under ice bath conditions, and the mixture was stirred for 12–24 h. Tetrahydrofuran was removed by vacuum distillation, and dichloromethane was added to ensure complete dissolution. The solution was filtered and then evaporated to dryness by rotary evaporation to obtain the crude product. Tetrahydrofuran was added dropwise while heating until just completely dissolved. The mixture was cooled to room temperature, and an equal volume of anhydrous diethyl ether was added. The mixture was allowed to stand overnight, resulting in the precipitation of white needle-like crystals, which was the trimethylene carbonate derivative monomer, with a yield of 58%.

[0063] Appendix Figure 1 The NMR spectrum is for a monomer of a trimethylene carbonate derivative. 1 H NMR (400MHz, CDCl3): -CH2-, δ = 4.11, 4.59ppm; -CH3, δ = 1.24ppm; -NH-C H 2-C H 2-NH-, δ=3.38ppm; C6H5-C H 2-, δ = 5.11ppm; C6H5-, δ = 5.26, 7.36ppm.

[0064] Example 2: Synthesis of an acid-sensitive, amphiphilic block polymer PEG-PTMC (Cit) modified with citrate anhydride

[0065] PEG-PTMC (CBZ) was synthesized via ring-opening polymerization using methoxy-terminated polyethylene glycol (mPEG-OH), trimethylene carbonate (TMC), and trimethylene carbonate derivative monomers as raw materials. Under argon protection, mPEG-OH, TMC, and trimethylene carbonate derivative monomers were added to a reaction flask, with 0.1% stannous octoate added as a catalyst. The polymerization reaction was carried out at 120°C for 24 h. After the reaction was completed, dichloromethane was added to dissolve the product, and excess cold diethyl ether was added to precipitate the product. The precipitate was collected by filtration and dried under vacuum to obtain the polymer intermediate PEG-PTMC (CBZ).

[0066] PEG-PTMC(CBZ) was dissolved in a mixed solvent of dichloromethane and methanol (9:1, v / v), and 10% Pd / C (w / v) was added. The mixture was purged with hydrogen three times and reacted at room temperature for 24 h with stirring. After the reaction was complete, Pd / C was removed by filtration, and tetrahydrofuran was removed by vacuum distillation of the filtrate. The filtrate was dissolved in a small amount of dichloromethane, and excess cold diethyl ether was added to precipitate the precipitate. The precipitate was collected by filtration and dried under vacuum to obtain PEG-PTMC(NH2).

[0067] 1 g of PEG-PTMC(NH2) was dissolved in anhydrous tetrahydrofuran, and citral anhydride (0.1–2 g) and pyridine (2 mL) were added. The reaction was carried out at room temperature for 24 h. After the reaction was completed, the solid byproducts were removed by filtration, and the tetrahydrofuran was removed by vacuum distillation of the filtrate. The crude product was dissolved in dimethyl sulfoxide, dialyzed to remove small molecule impurities, and lyophilized to obtain PEG-PTMC(Cit) products with different degrees of citral substitution.

[0068] In the synthesis of PEG-PTMC (CBZ), by controlling the reaction molar ratio of trimethylene carbonate derivative monomer to TMC to be 0:100, 5:95, 10:90, 20:80, 50:50, 75:25, and 100:0, copolymers of citonic acid with different degrees of substitution, namely PEG-PTMC, PEG-PTMC (5% Cit), PEG-PTMC (10% Cit), PEG-PTMC (20% Cit), PEG-PTMC (50% Cit), PEG-PTMC (75% Cit), and homopolymer PEG-PTMC (100% Cit), were obtained.

[0069] Appendix Figure 2 NMR spectrum of PEG-PTMC(Cit): 1 H NMR (400MHz, CDCl3):-OC H 2-C H 2-O-:δ=3.65ppm; -COO-C H 2-CH2-C H 2-O-: δ = 4.24ppm; -COO-CH2-CH 2-CH2-O-: δ = 2.06 ppm; -COO-C H 2-C-C H 2-O-: δ = 4.24 ppm; -CH3: δ = 1.25 ppm; -CH=C-C H 3: δ = 6.30, 6.48 ppm.

[0070] Example 3: Synthesis of succinic anhydride modified non-acid sensitive amphiphilic block polymer PEG-PTMC(SA)

[0071] PEG-PTMC(NH2) was first synthesized according to the method in Example 2. 1 g of PEG-PTMC(NH2) was dissolved in anhydrous tetrahydrofuran, succinic anhydride 1 g and 2 mL of pyridine were added, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the solid by-product was removed by filtration, and the filtrate was distilled under reduced pressure to remove tetrahydrofuran. The obtained crude product was dissolved in dimethyl sulfoxide, dialyzed to remove small molecular impurities, and freeze-dried to obtain the product PEG-PTMC(SA).

[0072] Figure 2 shows the1H NMR spectrum of PEG-PTMC(SA). Figure 3 The1H NMR spectrum of PEG-PTMC(SA) is shown in Figure 2. 1 H NMR (400 MHz, CDC13): -0-C H 2-C H 2-O-: δ = 3.65 ppm; -COO-C H 2-CH2-C H 2-O-: δ = 4.24 ppm; -COO-CH2-C H 2-CH2-O-: δ = 2.06 ppm; -COO-C H 2-C-C H 2-O-: δ = 4.24 ppm; -CH3: δ = 1.25 ppm; -C H 2-C H 2-COOH: δ = 2.52 ppm.

[0073] Example 4: Synthesis of 2,3-dimethyl maleic anhydride modified acid-sensitive responsive amphiphilic block polymer PEG-P(LA-co-TMC(DMA))

[0074]

[0075] PEG-P(LA-co-TMC(CBZ)) was synthesized by ring-opening polymerization reaction using mPEG-OH, lactide and trimethylene carbonate derivative monomer as raw materials. Specifically, mPEG-OH, lactide and trimethylene carbonate derivative monomer (molar ratio of trimethylene carbonate derivative monomer to lactide was 30:70) were added to a reaction flask under argon protection, and 0.1% stannous octoate was added as a catalyst, and the polymerization reaction was carried out at 120°C for 24h. After the reaction was completed, the product was dissolved in dichloromethane, and excess cold ether was added for precipitation. The precipitate was collected by filtration and vacuum dried to obtain the polymer intermediate PEG-P(LA-co-TMC(CBZ)).

[0076] 5g of PEG-P(LA-co-TMC(CBZ)) was dissolved in a mixed solvent of dichloromethane and methanol (volume ratio 9:1), 10% mass Pd / C was added, and hydrogen was replaced for 3 times. Under stirring conditions, the reaction was carried out at room temperature for 24h. After the reaction was completed, Pd / C was removed by filtration, and tetrahydrofuran was removed by vacuum distillation. A small amount of dichloromethane was added for dissolution, and excess cold ether was added for precipitation. The precipitate was collected by filtration and vacuum dried to obtain PEG-P(LA-co-TMC(NH2)).

[0077] 1g of PEG-P(LA-co-TMC(NH2)) was dissolved in 10mL of anhydrous tetrahydrofuran, 2,3-dimethyl maleic anhydride (2.5g) and 1mL of pyridine were added, and the reaction was carried out at room temperature for 24h. After the reaction was completed, the solid by-product was removed by filtration, and the filtrate was vacuum distilled to remove tetrahydrofuran. The obtained crude product was dissolved in dimethyl sulfoxide, and small molecular impurities were removed by dialysis. The product PEG-P(LA-co-TMC(DMA)) was obtained by freeze-drying with a yield of 90%.

[0078] Example 5: Synthesis of cis-aconitic anhydride modified acid-sensitive responsive amphiphilic block polymer PEG-P(CL-co-TMC(Aco))

[0079]

[0080] mPEG-OH, caprolactone and trimethylene carbonate derivative monomer were added to a reaction flask, wherein the molar ratio of trimethylene carbonate derivative monomer to caprolactone was 20:80, 50:50 and 80:20, respectively, and 0.1% stannous octoate was added as a catalyst. The polymerization reaction was carried out at 120°C for 24h under argon protection. After the reaction was completed, the product was dissolved in dichloromethane, and excess cold ether was added for precipitation. The precipitate was collected by filtration and vacuum dried to obtain the polymer intermediate PEG-P(CL-co-TMC(CBZ)).

[0081] PEG-P(CL-co-TMC(CBZ)) was dissolved in a mixed solvent of dichloromethane and methanol (volume ratio 9:1), 10% mass ratio of Pd / C was added, hydrogen was replaced for 3 times, and the reaction was carried out at room temperature for 24 h under stirring. After the reaction was completed, Pd / C was removed by filtration, the filtrate was distilled under reduced pressure to remove tetrahydrofuran, a small amount of dichloromethane was added for dissolution, and an excess of cold ether was added for precipitation. The precipitate was collected by filtration and dried under vacuum to obtain PEG-P(CL-co-TMC(NH2)).

[0082] PEG-P(CL-co-TMC(NH2)) was dissolved in 15 mL of anhydrous tetrahydrofuran, cis-aconitic anhydride (0.8-3 g) and 1 mL of pyridine were added, and the reaction was carried out at room temperature for 24-48 h. After the reaction was completed, the solid by-product was removed by filtration, and the filtrate was distilled under reduced pressure to remove tetrahydrofuran. The obtained crude product was dissolved in dimethyl sulfoxide, and small molecular impurities were removed by dialysis. PEG-P(CL-co-TMC(Aco)) was obtained by freeze-drying, with a yield of 87%-95%. At the same time, glutaric anhydride-modified non-acid-sensitive amphiphilic block polymer PEG-P(CL-co-TMC(GA)) was prepared. PEG-P(CL-co-TMC(NH2)) was dissolved in 15 mL of anhydrous tetrahydrofuran, glutaric anhydride (0.5-2 g) and 1 mL of pyridine were added, and the reaction was carried out at room temperature for 24-48 h. Impurities were removed by dialysis, and PEG-P(CL-co-TMC(GA)) was obtained by freeze-drying, with a yield of 91%-96%.

[0083] Example 6: Synthesis of 1-cyclohexene-1,2-dicarboxylic anhydride-modified acid-sensitive responsive amphiphilic block polymer PEG-P(LG-co-GA-co-TMC(DCA))

[0084]

[0085] mPEG-OH, lactide, glycolide and trimethylene carbonate derivative monomers were added to a reaction flask, the molar ratio of trimethylene carbonate derivative monomer to lactide and glycolide was 10:45:45, and 0.1% stannous octoate was added as a catalyst. The reaction was carried out under argon protection at 130°C for 24 h. After the reaction was completed, the product was dissolved in dichloromethane, and an excess of cold ether was added for precipitation. The precipitate was collected by filtration and dried under vacuum to obtain the polymer intermediate PEG-P(LG-co-GA-co-TMC(CBZ)).

[0086] 4 g of PEG-P(LG-co-GA-co-TMC(CBZ)) was dissolved in a mixed solvent of dichloromethane and methanol (9:1, volume ratio). 10% Pd / C was added, and the mixture was purged with hydrogen three times. The reaction was carried out at room temperature for 24 h under stirring. After the reaction was complete, Pd / C was removed by filtration, and tetrahydrofuran was removed by vacuum distillation of the filtrate. A small amount of dichloromethane was added to dissolve the precipitate, and excess cold diethyl ether was added to precipitate the precipitate. The precipitate was collected by filtration and dried under vacuum to obtain PEG-P(LG-co-GA-co-TMC(NH2)).

[0087] 3 g of PEG-P(LG-co-GA-co-TMC(NH2)) was dissolved in anhydrous tetrahydrofuran, and 1-cyclohexene-1,2-dicarboxylic anhydride (1-3 g) and 5 mL of pyridine were added. The reaction was carried out at room temperature for 24-48 h. After the reaction was completed, the solid byproducts were removed by filtration, and the tetrahydrofuran was removed by vacuum distillation of the filtrate. The crude product was dissolved in dimethyl sulfoxide, dialyzed to remove small molecule impurities, and lyophilized to obtain PEG-P(LG-co-GA-co-TMC(DCA)). In this step, 1-cyclohexene-1,2-dicarboxylic anhydride was replaced with the same molar amount of succinic anhydride, and the reaction was carried out in the same way to prepare the non-sensitive material PEG-P(LG-co-GA-co-TMC(SA)), with a yield of 87%. Meanwhile, in order to prepare materials labeled with the fluorescent molecule Cy5, PEG-P(LG-co-GA-co-TMC(NH2)) and Cy5-NHS active ester (0.05 Eq.) were first stirred and reacted for 6 h, and then reacted with the corresponding 1-cyclohexene-1,2-dicarboxylic anhydride or succinic anhydride. After dialysis purification, Cy5-PEG-P(LG-co-GA-co-TMC(DCA)) and Cy5-PEG-P(LG-co-GA-co-TMC(SA)) were obtained with yields of 83% and 80%, respectively.

[0088] Example 7: Preparation of PEG-PTMC(Cit) HN peptide nanoparticles with different degrees of citrate substitution

[0089] PEG-PTMC(5% Cit), PEG-PTMC(10% Cit), PEG-PTMC(20% Cit), PEG-PTMC(50% Cit), PEG-PTMC(75% Cit), PEG-PTMC(100% Cit) and unmodified PEG-PTMC prepared in Example 2 as the carrier material, the basic HN peptide-loaded nanoparticles were prepared by emulsion-solvent evaporation method: 20 mg of PEG-PTMC(Cit) or PEG-PTMC was accurately weighed and dissolved in 2 mL of dichloromethane. 500 μL of an aqueous solution containing 1 mg of HN peptide (sequence: MAPRGFSCLLLLTSEIDLPVKRRA) was accurately measured and added to the above PEG-PTMC(Cit) or PEG-PTMC dichloromethane solution. A probe-type ultrasonic cell pulverizer was used with a power of 100 W, ultrasonic treatment for 2 s, interval of 2 s, and working for 30 s to obtain a W / O type primary emulsion. 2 mL of a 2% sodium cholate aqueous solution was added to the primary emulsion, and intermittent ultrasonic treatment was performed at a power of 400 W for 30 s to obtain a W / O / W type multiple emulsion. The obtained multiple emulsion was added to 10 mL of a 0.5% sodium cholate aqueous solution, and dichloromethane was removed by rotary evaporation. The nanoparticle solution was placed in a high-speed refrigerated centrifuge and centrifuged at 4°C and 14000 rpm for 45 min. The supernatant was discarded, and the precipitate was dispersed with triple distilled water and washed three times to remove the unencapsulated free polypeptide drug. HPLC method was used for analysis and determination, and the encapsulation efficiency and drug loading of the nanoparticles were calculated according to the following formula.

[0090] Encapsulation efficiency = amount of encapsulated drug in nanoparticles / amount of drug administered x 100%

[0091] Drug loading = amount of encapsulated HN in nanoparticles / total weight of nanoparticles x 100%

[0092] The encapsulation efficiency and drug loading of HN peptide-loaded nanoparticles formed by six different substitution degrees of PEG-PTMC(Cit) are shown in Table 1. It can be seen that the encapsulation efficiency is greater than 35% and the drug loading is higher than 1.7%, and as the citraconic acid substitution degree increases from 5% to 100%, the encapsulation efficiency increases from 35% to 90%. Compared with the nanoparticles prepared by PEG-PTMC without carboxyl modification or PEG-PTMC(50% NH2) with amino groups in the side chain, the encapsulation efficiency (<10%) and drug loading (<1%) of HN peptide are very low. These results show that the introduction of multiple carboxyl groups in the side chain of PEG-PTMC material significantly enhances the encapsulation of basic HN peptide in nanoparticles due to the increase in electrostatic attraction.

[0093] Table 1 Encapsulation efficiency and drug loading of HN by PEG-PTMC(Cit) nanoparticles with different substitution degrees

[0094]

[0095] Example 8: Preparation of acid-sensitive polymer PEG-P(LA-co-TMC(DMA)) loaded with basic polypeptide nanoparticles

[0096] The PEG-P(LA-co-TMC(DMA)) prepared in Example 4 was used as the carrier material to prepare nanoparticles loaded with basic polypeptide by emulsion-solvent evaporation method: 25 mg of PEG-P(LA-co-TMC(DMA)) was precisely weighed and dissolved in 1 mL of chloroform. 100 μL of aqueous solution containing basic polypeptide (SS31, SAL or AGA) was precisely measured, and the polypeptide concentration was 20 mg / mL. The solution was added to the above PEG-P(LA-co-TMC(DMA)) chloroform solution. A probe-type ultrasonic cell pulverizer was used with a power of 100 W, ultrasonic 1 s, interval 1 s, and working 60 s to obtain a W / O type primary emulsion. 3 mL of 1% polyvinyl alcohol (PVA) aqueous solution was added to the primary emulsion, and intermittent ultrasonic was performed at a power of 300 W for 60 s to obtain a W / O / W type multiple emulsion. The obtained multiple emulsion was added to 30 mL of 0.25% PVA solution, and the chloroform was removed by rotary evaporation. The nanoparticle solution was placed in a high-speed refrigerated centrifuge and centrifuged at 4°C, 14000 rpm for 45 min. The supernatant was discarded, and the precipitate was dispersed with triple distilled water and washed three times to remove the unencapsulated free polypeptide drug. The encapsulation efficiency and drug loading of the nanoparticles were analyzed by HPLC method.

[0097] The results of the encapsulation efficiency and drug loading of PEG-P(LA-co-TMC(DMA)) forming nanoparticles loaded with three basic polypeptides are shown in Table 2. It can be seen that the encapsulation efficiency is greater than 60%, and the drug loading is higher than 5%.

[0098] Table 2 Encapsulation efficiency and drug loading of PEG-P(LA-co-TMC(DMA)) nanoparticles for three basic polypeptides

[0099]

[0100] Example 9: Preparation of PEG-P(CL-co-TMC(Aco)) and PEG-PLA mixed system loaded with NC-1900 peptide nanoparticles

[0101] PEG-P(CL-co-TMC(Aco)) or PEG-P(CL-co-TMC(GA)) prepared in Example 5 as the carrier material, mixed with PEG-PLA, and using the emulsion-solvent evaporation method to prepare the NC-1900 basic polypeptide-loaded nanoparticles: 21 mg of PEG-P(CL-co-TMC(Aco)) or PEG-P(CL-co-TMC(GA)) was precisely weighed and mixed with 9 mg of PEG-PLA, and 2 mL of dichloromethane was added to dissolve the materials. 200 μL of an aqueous solution containing 2.5 mg of NC-1900 polypeptide (sequence: Pyroglutomyl-NSPRG-NH2) was precisely measured and added to the dichloromethane solution of the above materials, and the probe was ultrasonicated at 200 W power for 2 s intervals for 60 s to obtain a W / O primary emulsion. 4 mL of a 1% PVA aqueous solution was added to the primary emulsion, and intermittent ultrasonication was performed at 300 W power for 60 s to obtain a W / O / W double emulsion. The obtained double emulsion was added to 20 mL of a 0.5% PVA aqueous solution, and dichloromethane was removed by rotary evaporation. The nanoparticle solution was centrifuged at 4°C and 14000 rpm for 45 min. The supernatant was discarded, and the precipitate was dispersed with triple distilled water and washed three times to remove the unencapsulated polypeptide drug.

[0102] The nanoparticles prepared in the PEG-P(CL-co-TMC(Aco)) and PEG-PLA mixed system had a particle size of 114.3 ± 5.3 nm, a PDI of 0.114 ± 0.008, a Zeta potential of -32.8 ± 3.8 mV, an encapsulation efficiency of 69.3 ± 4.1% for NC-1900 peptide, and a drug loading of 5.77 ± 0.34%. The nanoparticles prepared in the PEG-P(CL-co-TMC(GA)) and PEG-PLA mixed system had a particle size of 111.7 ± 3.3 nm, a PDI of 0.121 ± 0.014, a Zeta potential of -35.1 ± 2.7 mV, an encapsulation efficiency of 65.1 ± 3.2% for NC-1900 peptide, and a drug loading of 5.42 ± 0.26%.

[0103] Example 10: Preparation of Lysozyme-Loaded Nanoparticles in PEG-P(LA-co-TMC(DMA)) and PEG-PLGA Mixed System

[0104] Preparation of bFGF-loaded nanoparticles using PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PCL as carrier materials: 14 mg of PEG-P(LG-co-GA-co-TMC(DCA)) and 6 mg of PEG-PCL were precisely weighed and dissolved in 2 mL of dichloromethane. 200 μL of an aqueous solution containing 4 mg of bFGF was precisely measured and added to the dichloromethane solution of the above materials. An initial emulsion of W / O type was obtained by probe sonication at 200 W power for 2 s, with 2 s intervals for 20 s. 4 mL of a 1% aqueous solution of sodium cholate was added to the initial emulsion, and a re-emulsion of W / O / W type was obtained by intermittent sonication at 160 W power for 30 s. The obtained re-emulsion was added to 40 mL of a 0.2% aqueous solution of sodium cholate, and dichloromethane was removed by rotary evaporation. The nanoparticle solution was centrifuged at 14000 rpm for 45 min at 4°C. The supernatant was discarded, and the precipitate was dispersed with triple-distilled water and washed three times to remove unencapsulated bFGF.

[0105] The encapsulation efficiency of the nanoparticles prepared using the PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PCL mixed system for bFGF was 85.2 ± 2.9%, and the drug loading was 12.78 ± 0.43%. The particle size was 133.8 ± 3.2 nm, the PDI was 0.101 ± 0.021, and the Zeta potential was -48.7 ± 3.1 mV.

[0106] Example 11: Preparation of bFGF-loaded nanoparticles using PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PCL as carrier materials

[0107] Preparation of bFGF-loaded nanoparticles using PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PCL as carrier materials: 14 mg of PEG-P(LG-co-GA-co-TMC(DCA)) and 6 mg of PEG-PCL were precisely weighed and dissolved in 2 mL of dichloromethane. 200 μL of an aqueous solution containing 4 mg of bFGF was precisely measured and added to the dichloromethane solution of the above materials. An initial emulsion of W / O type was obtained by probe sonication at 200 W power for 2 s, with 2 s intervals for 20 s. 4 mL of a 1% aqueous solution of sodium cholate was added to the initial emulsion, and a re-emulsion of W / O / W type was obtained by intermittent sonication at 160 W power for 30 s. The obtained re-emulsion was added to 40 mL of a 0.2% aqueous solution of sodium cholate, and dichloromethane was removed by rotary evaporation. The nanoparticle solution was centrifuged at 14000 rpm for 45 min at 4°C. The supernatant was discarded, and the precipitate was dispersed with triple-distilled water and washed three times to remove unencapsulated bFGF.

[0108] The encapsulation efficiency of the nanoparticles prepared by the mixed system of PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PLGA for bFGF was 82.3±3.7%, and the drug loading was 16.5±0.74%. The particle size was 122.8±5.4 nm, the PDI was 0.124±0.058, and the Zeta potential was -33.7±2.8 mV.

[0109] Example 12: Preparation of PAG peptide-loaded polymer vesicles by the mixed system of PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PLGA

[0110] The PAG peptide-loaded polymer vesicles were prepared by the mixed system of PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PLGA prepared in Example 6 by the solvent injection method: 5 mg of PEG-P(LG-co-GA-co-TMC(DCA)) and 5 mg of PEG-PLGA and 1 mg of PAG peptide (sequence: PAG ASRLLLLTGEIDLP) were precisely weighed and dissolved in 4 mL of dimethyl sulfoxide, slowly injected into 20 mL of water, and stirred magnetically at room temperature for 6 h. Then, the polymer vesicle solution was transferred to an ultrafiltration tube and centrifuged at 4°C and 5000 rpm for 45 min. The ultrafiltrate was diluted with distilled water and washed three times to remove the organic solvent and unencapsulated polypeptide drug. The encapsulation efficiency and drug loading of the polymer vesicles were determined by HPLC.

[0111] The encapsulation efficiency of the polymer vesicles formed by PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-PLGA for basic PAG peptide was 77.9±4.1%, and the drug loading was 7.83±0.35%. The particle size was 123.6±2.4 nm, the PDI was 0.141±0.014, and the Zeta potential was -29.2±2.5 mV.

[0112] Example 13: Acid-sensitive response characteristics of nanoparticles prepared by the polymers PEG-P(CL-co-TMC(Aco)) and PEG-P(CL-co-TMC(GA))

[0113] Twenty-five mg of NC-1900 peptide-loaded PEG-P(CL-co-TMC(Aco)) and PEG-P(CL-co-TMC(GA)) nanoparticles prepared in Example 9 were mixed with 20 mL of pH 7.4 PBS and 20 mL of pH 5.0 phosphate buffer and placed at 37°C. Three samples were taken at 0, 1, 4, and 12 h, respectively, diluted with 10 times the volume of water, and the Zeta potential of the nanoparticles was measured.

[0114] The zeta potential changes of PEG-P(CL-co-TMC(Aco)) and PEG-P(CL-co-TMC(GA)) nanoparticles in two pH buffers are shown in the attached figure. Figure 4 As shown, PEG-P(CL-co-TMC(Aco)) nanoparticles, after incubation in pH 7.4 buffer for 12 h, exhibited minimal changes in zeta potential, remaining within the range of -27 to -33 mV. However, after incubation at pH 5.0, their zeta potential showed a significant change, rising to +8 mV after 4 h and further increasing to +13.5 mV after 12 h, reversing from negative to positive, demonstrating obvious acid sensitivity. In contrast, the zeta potential of PEG-P(CL-co-TMC(GA)) nanoparticles remained almost unchanged in both pH buffers, indicating that they do not possess acid-sensitive properties.

[0115] 25 mg of PEG-P (CL-co-TMC(Aco)) or PEG-P (CL-co-TMC(GA)) nanoparticles loaded with NC-1900 peptide were uniformly dispersed in 10 mL of pH 7.4 PBS and 10 mL of pH 5.0 phosphate buffer. After mixing, the mixture was divided into 24 aliquots and placed in a shaker (37℃, 100 rpm). Three aliquots were taken at 0, 0.5, 1, 2, 4, 8, 12, and 24 h, respectively, and immediately centrifuged at 4℃, 14000 rpm for 45 min, and the supernatant was discarded. The content of NC-1900 retained in the nanoparticles at 0 h and each time point after release was determined by HPLC (denoted as C0 and C10, respectively). t The cumulative release percentage (CR) of the nanoparticles is calculated using the following formula. t ):

[0116] CR t (%) = (C0 - C) t ) / C0*100%

[0117] With CR t Plot the release curve against time t.

[0118] The in vitro release curves of PEG-P(CL-co-TMC(Aco)) and PEG-P(CL-co-TMC(GA)) nanoparticles are attached. Figure 5PEG-P(CL-co-TMC(Aco)) nanoparticles showed a significant difference in release in the two media, showing a slow and incomplete release behavior at pH 7.4, while the drug release was significantly accelerated in the pH 5.0 medium, with a 24 h cumulative release percentage of 89.2%. The release behavior of PEG-P(CL-co-TMC(GA)) nanoparticles in the two pH buffers was similar, with a release of less than 45% within 24 h.

[0119] The above results show that the nanoparticles prepared from the acid-sensitive material PEG-P(CL-co-TMC(Aco)) exhibit obvious Zeta potential reversal and accelerated release characteristics under low pH conditions.

[0120] Example 14: Lysosomal escape ability of nanoparticles prepared from the acid-sensitive polymer PEG-P(LG-co-GA-co-TMC(DCA))

[0121] PEG-P(LG-co-GA-co-TMC(DCA)) and PEG-P(LG-co-GA-co-TMC(SA)) synthesized in Example 6 were used to prepare FITC-labeled SAL peptide-loaded nanoparticles by the emulsion-solvent evaporation method described in Example 8.

[0122] Hippocampal neuron cells HT22 were seeded at a density of 1 x 10 4 / cm 2 in a confocal dish and cultured in a constant temperature incubator for 24 h. The culture solution was aspirated, the cells were incubated with PBS for 15 min, then 50 μg / mL of FITC-labeled SAL-loaded nanoparticles were added, and the cells were incubated at 37°C for 2 h. The nanoparticle solution was aspirated, serum-free DMEM medium was added, and the cells were further incubated for 0, 2, 6, and 10 h, respectively, and then washed with cold PBS 3 times. 50 nM LysoTracker Red DND-99 (lysosome dye) diluted in DMEM was added and incubated for 30 min, washed with PBS 3 times, then 10 μg / mL Hoechst 33342 (nuclear dye) was added and incubated for 10 min, washed with PBS 3 times, and observed and photographed under a laser confocal microscope. TM

[0123] The relative positions of the nanoparticles (green) and lysosomes (red) were observed under a laser confocal microscope, as shown in FIG. 6. The results show that the nanoparticles were mainly localized in the cytoplasm of the cells, and the FITC-labeled SAL peptide was released from the nanoparticles and entered the lysosomes, which is consistent with the results of the in vitro release experiment. Figure 6 ​As shown, after 2h incubation with HT22 cells, the green fluorescence of the nanoparticles was obviously coincided with the red fluorescence of lysosomes, showing yellow color, indicating that after the nanoparticles were taken up by the cells, a large amount of them entered lysosomes. When the incubation time was prolonged to 10h, the green fluorescence of the PEG-P(LG-co-GA-co-TMC(SA)) nanoparticles was still obviously coincided with the red fluorescence of lysosomes, indicating that the acid-insensitive nanoparticles were difficult to escape from lysosomes. While the PEG-P(LG-co-GA-co-TMC(DCA)) nanoparticles gradually reduced the co-localization with lysosomes as the incubation time was prolonged, and there was basically no coincidence between the green and red fluorescence at 10h. The co-localization coefficient R of the green and red fluorescence was calculated by Image J software (R = 0-0.6, no co-localization; R = 0.6-1, co-localization), and the change trend of R value was consistent with the above observation results. It was shown that after the PEG-P(LG-co-GA-co-TMC(DCA)) nanoparticles were taken up by HT22 into lysosomes, they could undergo charge reversal to carry more positive charges, thus accelerating the escape from lysosomes.

[0124] Example 15: Intracellular drug release of nanoparticles prepared from acid-sensitive polymer PEG-P(LG-co-GA-co-TMC(DCA))

[0125] Cy5-PEG-P(LG-co-GA-co-TMC(DCA)) and Cy5-PEG-P(LG-co-GA-co-TMC(SA)) synthesized in Example 6 were used to prepare FITC-SAL-loaded nanoparticles by emulsification-solvent evaporation method as described in Example 8. The obtained double-fluorescently labeled nanoparticles were recorded as Cy5-NP(DCA) / FITC-SAL and Cy5-NP(SA) / FITC-SAL, respectively.

[0126] HT22 cells were seeded at a density of 1 x 10 4 / cm 2 in confocal dishes and cultured in a constant temperature incubator for 24h. After the culture solution was aspirated, the cells were incubated with PBS for 15min, and then 50μg / mL double-fluorescently labeled nanoparticles were added, followed by incubation at 37℃ for 2h. The nanoparticle solution was aspirated, and serum-free DMEM medium was added for further incubation for 0 and 6h, respectively. The cells were washed with cold PBS for 3 times. Then 10μg / mL Hoechst 33342 (nuclear dye) was added for incubation for 10min, and the cells were washed with PBS for 3 times. The cells were observed and photographed under a laser confocal microscope.

[0127] The relative position of the carrier and the polypeptide in the cell is shown in the accompanying drawings Figure 7As shown in Figure 6, the results show that after incubation of Cy5-NP(DCA) / FITC-SAL nanoparticles with cells for 6 h, the red fluorescence of the intracellular nanocarriers and the green fluorescence of the polypeptide are separated, indicating that FITC-SAL is basically completely released from the nanoparticles. After incubation of non-acid-sensitive Cy5-NP(SA) / FITC-SAL nanoparticles for 6 h, the red and green fluorescence still significantly overlap, proving that the nanoparticles do not release FITC-SAL. The above results prove that the nanoparticles prepared from the acid-sensitive material PEG-P(LG-co-GA-co-TMC(DCA)) have a faster intracellular drug release capacity.

[0128] Example 16: Evaluation of the drug-loaded nanoparticles prepared from PEG-PTMC(10%Cit) for improving cell tolerance to Aβ toxicity

[0129] PEG-PTMC(10%Cit) and PEG-PTMC(10%SA) synthesized in Examples 2 and 3 were used to prepare AGA peptide-loaded nanoparticles by the emulsification-solvent evaporation method described in Example 7, and were named NP(Cit) and NP(SA), respectively.

[0130] HT22 cells were seeded at a density of 5 x 10 4 / cm 2 in a 96-well cell culture plate and cultured in an incubator for 24 h. The culture solution was aspirated, and the cells were washed with PBS for 15 min. Each group was treated as follows: normal control group: serum-free medium was added and incubated for 36 h; Aβ group: after incubation with serum-free DMEM for 12 h, 25 μM Aβ 25-35 solution was added to each well, and incubation was continued for 24 h; drug administration group: first incubation with AGA solution diluted with serum-free DMEM, NP(Cit) and NP(SA) (AGA concentration was 10 μM) for 12 h, and then 25 μM Aβ 25-35 was added, and incubation was continued for 24 h. After incubation, the sample solution was aspirated, washed with PBS for 3 times, 100 μL of 1 mg / mL MTT solution was added, and incubation was continued at 37°C for 4 h. The MTT solution was discarded, 150 μL of DMSO was added and shaken thoroughly to dissolve the purple formazan, and the absorbance value was determined at 490 nm using a microplate reader. The experimental results are shown in Figure 7. Figure 8 As shown in Figure 7, Aβ 25-35 has cytotoxicity, and can reduce the viability of HT22 cells to 53%. After treatment with AGA solution or NP(SA), both can partially resist the toxicity of Aβ 25-35 , but the cell viability is still lower, which is 62% and 71% of the normal control group, respectively; and NP(Cit) has a significant improvement effect, and the cell viability after treatment is increased to 93.4% of the normal control group.

[0131] The polypeptide-loaded nanoparticles prepared by using the acid-sensitive polymer PEG-PTMC (10% Cit) as the material can escape from the lysosome more quickly, and release the drug sufficiently into the cytoplasm of the cell, thereby better exerting the anti-Aβ effect of the AGA peptide 25-35 The cytotoxicity is reduced, and the drug efficacy is significantly improved.

[0132] Example 17: Nanoparticles prepared by using the polymer PEG-P(LA-co-TMC(DMA)) improve the stability of the loaded SAL peptide

[0133] 100 μL of the SAL peptide-loaded PEG-P(LA-co-TMC(DMA)) nanoparticles and SAL solution prepared in Example 8 were respectively mixed with 1 mL of mouse blank plasma or brain tissue homogenate, and incubated in a 37℃ shaking incubator (100 rpm). The SAL concentration in the samples was determined at 0.5, 1, 2, 4 and 8 h, and the content change relative to 0 h was calculated.

[0134] The SAL concentration change after the incubation of the SAL solution and the SAL-loaded nanoparticles with the plasma and the brain tissue homogenate for different time is shown in Table 1. Figure 9 As shown in Table 1, the free SAL peptide is easily degraded in the plasma and the brain tissue homogenate. After 8 h of incubation with the plasma, the content is reduced to 29% of the initial concentration; and after 8 h of incubation with the brain tissue homogenate, the content is reduced to 11% of the initial concentration. However, after 8 h of incubation of the SAL peptide-loaded nanoparticles with the plasma and the brain tissue homogenate, 75% and 47% of the SAL is still retained, indicating that the nanoparticle loading effectively improves the stability of the SAL peptide in the blood and the brain tissue.

[0135] Example 18: MTT method for evaluating the cytotoxicity of PEG-P(LA-co-TMC(DMA)), PEG-P(CL-co-TMC(Aco)) and PEG-P(LG-co-GA-co-TMC(DCA)) nanoparticles

[0136] The hippocampal neuron cells HT22 and the brain capillary endothelial cells bEnd.3 were respectively seeded in 96-well plates at a density of 5×10 4 / cm 2The density was inoculated in 96-well cell culture plates, and incubated in a thermostat incubator for 24 h. The culture solution was aspirated, and washed with PBS. The blank nanoparticles prepared by PEG-P(LA-co-TMC(DMA)), PEG-P(CL-co-TMC(Aco)), and PEG-P(LG-co-GA-co-TMC(DCA)) were diluted to concentrations of 50, 100, 500, 1000, and 2000 μg / mL, respectively, and added to the culture plates, and incubated at 37 °C for 24 h. After the incubation, the sample solution was aspirated, washed with PBS for 3 times, 100 μL of 1 mg / mL MTT solution was added, and incubated at 37 °C for 4 h. The MTT solution was discarded, 150 μL of DMSO was added and shaken well to dissolve the generated purple formazan. The absorbance value was measured at 490 nm by using a microplate reader. The experimental results are shown in FIG. 3, and the three kinds of nanoparticles had no cytotoxicity at concentrations of 50-2000 μg / mL, and the survival rates of the two cells were both >80%, indicating that the PEG-P(LA-co-TMC(DMA)), PEG-P(CL-co-TMC(Aco)), and PEG-P(LG-co-GA-co-TMC(DCA)) materials synthesized in the application had good biological safety. Figure 10 The experimental results are shown in FIG. 3, and the three kinds of nanoparticles had no cytotoxicity at concentrations of 50-2000 μg / mL, and the survival rates of the two cells were both >80%, indicating that the PEG-P(LA-co-TMC(DMA)), PEG-P(CL-co-TMC(Aco)), and PEG-P(LG-co-GA-co-TMC(DCA)) materials synthesized in the application had good biological safety.

Claims

1. An acid-sensitive polymer containing β-carboxylamide linkages, characterized in that: The polymer is an acid-sensitive responsive amphiphilic block polymer containing β-carboxyl amide bond in side chain, wherein the hydrophilic segment is polyethylene glycol (PEG), and the hydrophobic segment is a copolymer based on trimethylene carbonate derivative, The chemical structure is as follows: R1 is selected from one of the following groups: R2 is selected from one of the following groups: In the structural formula, n is 20-120, x is 50-500, y is 25-250, and the ratio of x to y is 20:1-1:

5.

2. The β-carboxamide group-containing acid-sensitive polymer according to claim 1, characterized by: The β-carboxyl amide bond is hydrolyzed and broken under acidic conditions of pH 4-6.8; the anhydride used to form the β-carboxyl amide bond is selected from citraconic anhydride, 2,3-dimethyl maleic anhydride, cis-aconitic anhydride, and 1-cyclohexene-1,2-dicarboxylic anhydride.

3. The β-carboxamide group-containing acid-sensitive polymer according to claim 1, characterized by: The copolymer of the trimethylene carbonate derivative is copolymerized from trimethylene carbonate derivative monomers and other carbonate or cyclic ester monomers; The chemical structure of the trimethylene carbonate derivative monomer is as follows:

4. The acid-sensitive polymer containing β-carboxyl amide linkage according to claim 3, characterized in that: The carbonate monomers participating in copolymerization are selected from trimethylene carbonate, 2,2-dimethyl trimethylene carbonate.

5. The β-carboxamide group-containing, acid-sensitive polymer according to claim 3, characterized by: The cyclic ester monomers participating in copolymerization are selected from lactide, glycolide, and caprolactone.

6. The β-carboxamide bond-containing, acid-sensitive polymer of claim 1, wherein: The synthesis includes the following steps: (1) Using polyethylene glycol as an initiator, trimethylene carbonate derivative monomers and other carbonate or cyclic ester monomers are subjected to ring-opening copolymerization to prepare random copolymer intermediate a, and the chemical structure of the copolymer intermediate a is as follows: R1 is selected from one of the following groups: (2) The copolymer intermediate a is deprotected under Pd / C / H2 conditions to obtain copolymer intermediate b, and the side chain of the copolymer intermediate b has a primary amine group, and the chemical structure of the copolymer intermediate b is as follows: (3) The primary amine group of the copolymer intermediate b side chain reacts with anhydride to form a β-carboxyl amide bond to obtain an acid-sensitive responsive amphiphilic block polymer, and the chemical structure of the acid-sensitive responsive amphiphilic block polymer is as follows: R2 is selected from one of the following groups:

7. The β-carboxamide bond-containing, acid-sensitive polymer according to claim 3, characterized in that: The synthesis of the trimethylene carbonate derivative monomer includes the following steps: (1) 2,2-bis(hydroxymethyl)propionic acid and bis(pentafluorophenyl) carbonate are reacted to prepare intermediate 1, and the chemical structure of the intermediate 1 is as follows: (2) The intermediate 1 is reacted with N-benzyloxy carbonyl ethylenediamine to obtain the trimethylene carbonate derivative monomer.

8. The acid-sensitive polymer containing β-carboxyl amide linkages according to claim 7, characterized in that: In the synthesis of the trimethylene carbonate derivative monomer, the molar ratio of 2,2-bis(hydroxymethyl)propionic acid to bis(pentafluorophenyl) carbonate is 1:2-1:5, and the molar ratio of intermediate 1 to N-benzyloxy carbonyl ethylenediamine is 3:1-1:

3.

9. Use of the acid-sensitive polymer containing β-carboxyl amide bond according to claim 1 in the preparation of a drug carrier, wherein the drug carrier is suitable for loading basic polypeptide / protein drugs, the encapsulation efficiency is 35%-90%, and the drug loading capacity is 1%-17%.

10. Use according to claim 9, characterized in that: The form of the drug carrier is selected from polymer nanoparticles, polymer vesicles, polymer micelles, and nanocomposites.

11. Use according to claim 9, characterized in that: The drug carrier is prepared by mixing the acid-sensitive responsive amphiphilic block polymer with other synthetic amphiphilic polymers, wherein the mass ratio of the acid-sensitive responsive block polymer is 50% to 100%, and the other synthetic amphiphilic polymers are selected from polyethylene glycol-polytrimethylene carbonate, polyethylene glycol-poly-lactic acid, polyethylene glycol-poly(lactic acid-glycolic acid) copolymer, polyethylene glycol-poly-caprolactone, polyethylene glycol-poly-hydroxybutyric acid ester, polyethylene glycol-poly-glyceryl sebacate, and polyethylene glycol-poly-citric acid.

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